The present invention is in the field of immunology. In particular, this invention is directed to methods of immunization using compositions comprising cationic lipids and polynucleotide molecules which code for immunogens. This invention is also directed to methods for producing polyclonal and monoclonal antibodies from genetically immunized animals. This invention is further directed to the use of genetic immunization to map protein epitopes.
Traditional methods of immunization are achieved by injection of a mixture of antibodies which immunoreact with an invading pathogen (i.e., passive immunization), or by vaccination, which stimulates the immune system to produce pathogen-specific antibodies. Since foreign antibodies are cleared by the recipient, passive immunity confers only temporary protection. Vaccination confers longer-lasting active immunity.
In order to be effective, vaccination must generate humoral and/or cell-mediated immunity which will prevent the development of disease upon subsequent exposure to the corresponding pathogen. The pertinent antigenic determinants must be presented to the immune system in a manner that mimics a natural infection. Conventional viral vaccines may consist of inactivated virulent strains, or live-attenuated strains (Old et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering, Blackwell Scientific Publications, 4th edition, 1989). A general problem with using a vaccine consisting of a virus is that many viruses (such as hepatitis B virus) have not been adapted to grow to high titre in tissue culture and thus, cannot be produced in sufficient quantity (Id.). In addition, the use of inactivated viruses present a potential danger of vaccine-related disease resulting from replication-competent virus may remain in the inoculum. Outbreaks of foot-and-mouth disease in Europe have been attributed to this cause (Id.). On the other hand, attenuated virus strains have the potential to revert to virulent phenotype upon replication in the vaccinee. This problem has been reported to occur about once or twice in every million people who receive live polio vaccine (Id.). Moreover, encephalitis can occur following measles immunization with attenuated virus (Roit, I. M. Essential Immunology, Blackwell Scientific Publications, Sixth Ed., 1988). Another disadvantage of using attenuated strains is the difficulty and expense of maintaining appropriate cold storage facilities (Id.). A major disadvantage associated with the use of live virus vaccines is that persons with congenital or acquired immunodeficiency risk severe infections. Such persons include children in developing countries who are often immunodeficient because of malnutrition and/or infection with viruses or parasites (Id., Old et al., supra).
As a result of recent advances in molecular biology and peptide synthesis, it is possible to produce purified viral proteins or synthetic peptides for use in immunoprophylaxis (Murphy et al., “Immunization Against Viruses,” in Virology, Fields et al., Eds., Raven Press, New York, pp. 349–370, 1985). Purified antigens may be produced by synthesizing peptides which represent immunologically important domains of surface antigens of the pathogen. The synthetic peptide approach has been successfully used with an antigenic determinant of the foot and mouth disease virus (Id.). One problem with this approach is that the poor antigenicity of synthetic peptides has required the use of Freund's adjuvant to enhance the immune response in experimental animals (Id.). Since Freund's adjuvant cannot be used in humans, an effective adjuvant for human use must be developed (Id.). In addition, a single antigenic site may not be sufficient to induce resistance since large surface antigens usually contain several distinct immunological domains that elicit a protective humoral and/or cell-mediated response (Braciale et al., J. Exp. Med. 153:910–923 (1981); Wiley et al., Nature 289:373–378 (1981)). There may also be difficulties in stimulating an immunologic response to epitopes that are formed by noncontiguous parts of the linear protein molecule (Murphy, et al., supra). There is evidence that the majority of protein determinants are discontinuous and involve amino acid residues that are far apart in the primary amino acid sequence, but are brought into close juxtaposition by peptide folding (Roit, supra).
The alternative approach to preparing proteins for vaccines involves the use of cloned viral DNA inserted into a suitable vector to produce viral protein in prokaryotic or eukaryotic cells (Aldovini et al., The New Vaccines, Technology Review, pp. 24–31, January 1992). This approach, also, has several limitations. For example, one must devise suitable conditions for the optimal production of the recombinant protein of interest by the recombinant host cells. The protein product must be isolated and purified from the culture system, and obtained in sufficient quantities for use as a vaccine. Finally, it may be necessary to perform post-translational modifications of the purified protein (such as glycosylation and/or cleavage of a fusion protein).
An alternative to producing the recombinant antigen in vitro is to introduce nucleic acid sequences coding for the antigen into the cells of the vaccinee. In this way, the antigen is produced in vivo by the vaccinee's cells and provokes the immune response. Tang et al. (Nature 356:152–154 (1992)) have shown that it is possible produce an immune response to human growth hormone protein in mice by propelling gold microprojectiles coated with plasmids containing human growth hormone genomic sequences. The resultant variability in the production of antibody production was hypothesized to arise from the operation of the microprojectile device, or the coating of the DNA onto the microprojectiles.
More recently, Ulmer et al. (Science 259:1745–1749 (1993)) injected a plasmid carrying the gene for influenza A nucleoprotein into the quadriceps of mice. The mice produced nucleoprotein antibodies, indicating that the gene was expressed in murine cells. The mice also produced nucleoprotein-specific cytotoxic T lymphocytes which were effective in protecting the mice from a subsequent challenge with a heterologous strain of influenza A virus. Similarly, Wang et al. (Proc. Natl. Acad. Sci. USA 90:4156–4160 (1993)) observed that the intramuscular injection of a human immunodeficiency virus (HIV) type 1 envelope DNA construct in mice generated antigen-specific cellular and humoral immune responses. In addition, splenic lymphocytes derived from the inoculated mice demonstrated HIV-envelope-specific proliferative responses. Thus, direct inoculation of DNA coding for pathogenic antigens can provide an alternative to the use of viruses, proteins, or peptides.
One problem with using naked DNA for inoculation is the low efficiency of cellular uptake. For example, the protocol of Wang et al., supra, requires the injection of 100 micrograms of the DNA construct biweekly for a total of four inoculations. As described herein, the use of cationic lipids as a carrier for DNA constructs provides a more efficient means of genetic immunization. According to the present invention, genetic immunization can be achieved with as little as 5 micrograms of a DNA construct, which has been complexed with cationic lipid.
Liposomes have been used as carriers of genetic information in the transfection of tissue culture cells. A fundamental problem of liposome-mediated transfection with liposomes comprising neutral or anionic lipids is that such liposomes do not generally fuse with the target cell surface. Instead, the liposomes are taken up phagocytically, and the polynucleotides are subsequently subjected to the degradative enzymes of the lysosomal compartment (Straubinger et al., Methods Enzymol. 101:512–527 (1983); Mannino et al., Biotechniques 6:682–690 (1988)). Another problem with conventional liposome technology is that the aqueous space of typical liposomes may be too small to accommodate large macromolecules such as DNA or RNA. As a result, typical liposomes have a low capturing efficiency (Felgner, “Cationic Liposome-Mediated Transfection with Lipofectin™ Reagent,” in Gene Transfer and Expression Protocols Vol. 7, Murray, E. J., Ed., Humana Press, New Jersey, pp. 81–89 (1991)).
Liposomes comprising cationic lipids interact spontaneously and rapidly with polyanions such as DNA and RNA, resulting in liposome/nucleic acid complexes that capture 100% of the polynucleotide (Felgner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413–7417 (1987); Felgner et al., Focus 11:21–25 (1989)). Moreover, the polycationic complexes are taken up by the anionic surface of tissue culture cells with an efficiency that is about ten to one hundred times greater than negatively charged or neutral liposomes (Felgner, “Cationic Liposome-Mediated Transfection with Lipofectin™ Reagent,” in Gene Transfer and Expression Protocols Vol. 7, Murray, E. J., Ed., Humana Press, New Jersey, pp. 81–89 (1991)). In addition, the polycationic complexes fuse with cell membranes, resulting in an intracellular delivery of polynucleotide that bypasses the degradative enzymes of the lysosomal compartment (Duzgunes et al., Biochemistry 28:9179–9184 (1989); Felgner et al., Nature 337:387–388 (1989)).
Various formulations of cationic lipids have been used to transfect cells in vitro (WO 91/17424; WO 91/16024; U.S. Pat. Nos. 4,897,355; 4,946,787; 5,049,386; and 5,208,036). Cationic lipids have also been used to introduce foreign polynucleotides into frog and rat cells in vivo (Holt et al., Neuron 4:203–214 (1990); Hazinski et al., Am. J. Respr. Cell. Mol. Biol. 4: 206–209 (1991)). Therefore, cationic lipids may be used, generally, as pharmaceutical carriers to provide biologically active substances (for example, see WO 91/17424; WO 91/16024; and WO 93/03709). Thus, cationic liposomes can provide an efficient carrier for the introduction of foreign polynucleotides into host cells for genetic immunization.
Various cationic lipids are well-known in the prior art. One well-known cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). The structure of DOTMA is:

DOTMA, alone or in a 1:1 combination with dioleoylphosphatidylethanolamine (DOPE) can be formulated into liposomes using standard techniques. Felgner et al. (Proc. Natl. Acad. Sci. U.S.A. 84:7413–7417 (1987)) have shown that such liposomes provide efficient delivery of nucleic acids to cultured cells. A DOTMA:DOPE (1:1) formulation is sold under the name LIPOFECTIN™ (GIBCO/BRL: Life Technologies, Inc., Gaithersburg, Md.). Another commercially available cationic lipid is 1,2-bis(oleoyloxy)-3-3-(trimethylammonia)propane (DOTAP), which differs from DOTMA in that the oleoyl moieties are linked via ester bonds, not ether bonds, to the propylamine. DOTAP is believed to be more readily degraded by target cells.
A related groups of known compounds differ from DOTMA and DOTAP in that one of the methyl groups of the trimethylammonium group is replaced by a hydroxyethyl group. Compounds of this type are similar to the Rosenthal Inhibitor of phospholipase A (Rosenthal et al., J. Biol. Chem. 235:2202–2206 (1960), which has stearoyl esters linked to the propylamine core. The dioleoyl analogs of the Rosenthal Inhibitor (RI) are commonly abbreviated as DORI-ether and DORI-ester, depending upon the linkage of the fatty acid moieties to the propylamine core. The hydroxy group can be used as a site for further functionalization, for example, by esterification to carboxyspermine.
Another class of known compounds has been described by Behr et al. (Proc. Natl. Acad. Sci. USA 86:6982–6986 (1989); EPO Publication 0 394 111), in which carboxyspermine has been conjugated to two types of lipids. The structure of 5-carboxylspermylglycine dioctadecylamide (DOGS) is:

The structure of dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DDPES) is:

Both DOGS and DPPES have been used to coat plasmids, forming a lipid aggregate complex that provides efficient transfection. The compounds are claimed to be more efficient and less toxic than DOTMA for transfection of certain cell lines. DOGS is available commercially as TRANSFECTAM™ (Promega, Madison, Wis.).
A cationic cholesterol derivative (DC-Chol) has been synthesized and formulated into liposomes in combination with DOPE (Gao et al., Biochim. Biophys. Res. Comm. 179:280–285 (1991)). The structure of this compound is:

Liposomes formulated with DC-Chol provide more efficient transfection and lower toxicity than DOTMA-containing liposomes for certain cell lines.
Lipopolylysine is formed by conjugating polylysine to DOPE. This compound has been reported to be especially effective for transfection in the presence of serum (Zhou et al., Biochim. Biophys. Res. Comm. 165:8–14 (1991)). Thus, lipopolylysine may be an effective carrier for immunization.
In addition, Gebeyhu et al. (co-pending U.S. application Ser. No. 07/937,508; filed Aug. 28, 1992) have developed novel cationic lipids according to the general formula:
wherein R1 and R2 separately or together are C1-23 alkyl or
    alkyl or alkenyl, q is 1 to 6,    Z1 and Z2 separately or together are H or unbranched alkyl C1-6     X1 is —(CH2)nBr, Cl, F or I, n=0–6 or    X2 is —(CH2)nNH2 n=0–6 or    X3 is —NH—(CH2)m—NH2 m=2–6 or    X4 is —NH—(CH2)3—NH—(CH2)4—NH2 or    X5 is —NH—(CH2)3—NH—(CH2)4—NH(CH2)3—NH2     X6 is
    X7 is
    X8 is
    where p is 2–5, Y is H or other groups attached by amide or alkyl amino group or    X9 is a polyamine, e.g., polylysine, polyarginine, polybrene, histone or protamine or    X10 is a reporter molecule, e.g., biotin, folic acid or PPD, or
    X11 is a polysaccharide or substituted polysaccharide, or    X12 is a protein or    X13 is an antibody or    X14 is an amine or halide reactive group or    X15 is —(CH2)r—SH where r is 0–6 or    X16 is —(CH2)s—S—S—(CH2)t—NH2 where s is 0–6 and t is 2–6.
These compounds are useful either alone, or in combination with other lipid aggregate-forming components (such as DOPE or cholesterol) for formulation into liposomes or other lipid aggregates. Such aggregates are cationic and able to complex with anionic macromolecules such as DNA or RNA.